Fig. 4: DNA shuffling dissects the roles of polymorphisms within the LRR20-EJM region of GmFLS2a/b and generates greater recognition capacity towards flg22Rso.
a, Schematic diagram of the DNA shuffling workflow. See Methods section for detailed explanations. For illustrative purposes only, the numbers on the leaves indicate the serial numbers of the shuffled variants tested, while the dashed lines mark the infiltrated regions. b, Summary of the sequences and performances of 30 shuffled variants. c, Analysis of DNA shuffling results. See the Methods section for detailed explanations of this plot. d, Flg22Rso-induced ROS production of GmFLS2a with single and double mutations of H600Q and S603A (n = 12 leaf discs). e, Effects of A603S and H600Q on the flg22Rso-induced ROS response of chimera aaba (n = 12 leaf discs). f, Effects of DNA shuffling-predicted enhancer and ‘suppressor’ polymorphisms on flg22Rso-induced ROS production (n = 36, 80, 36, 44, 36 leaf discs, from top to bottom). See c for the definitions of E, P1, P2, P3 and N. g, Comparison of total flg22Rso-induced ROS production between GmFLS2b and SuperGmFLS2 (n = 138 and 130 leaf discs, from left to right). SuperGmFLS2 combines all the GmFLS2b-originated (orange) regions/polymorphic residues with positive effects on flg22Rso recognition. h, Co-IP of NbBAK1 with wild-type GmFLS2a and two engineered variants. For bar plots and line charts, data are presented as mean. For box plots: centre line, median; box limits, upper and lower quartiles; whiskers, 1.5 times interquartile range; points, outliers; ‘+’, mean. Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used for statistical analysis in d–f with significant differences at P < 0.05 indicated by letters. See Source data for exact P values. Two-tailed Mann–Whitney test was used for g. ROS experiments in d–g were repeated three times with similar results. Co-IP experiment in h was repeated twice with similar results. Panel a created with BioRender.com.
